Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. We propose to define critical aspects of factor VIII structure and regulation that includes fine point details of inter-subunit interactions, the role of calcium in stabilizing factor VIII structure and mechanisms for the activation of factor VIII by thrombin. Aim I focuses on interactions of A2 subunit with the A1/A3C1C2 dimer in the factor VIIIa heterotrimer. Specific studies will elucidate mechanisms for the retention of A2 subunit in factor VIIIa, expanding on our prior work that mapped a number of hydrogen bonds to the interface of A2 with A1 and A3 domains and identified high stability factor VIIIa variants resulting from removal of destabilizing charged residues from hydrophobic pockets. The identification of several residues that appear functionally important in bonding interactions in factor VIIIa but not factor VIII suggests sites for structural changes resulting from procofactor activation. These sites will be characterized regarding inter-subunit affinity parameters and identification of potential bonding partners. Aim II studies focus on the functional role of a Ca2+ site we previously identified in the factor VIII A1 domain. Mutagenesis experiments are proposed to enhance stability at this site by altering noncovalent interactions in an effort to recapitulate our recent work stabilizing this region by covalent bonding. This study includes generation of gain-of-function variants possessing increased specific activity coupled with increased stability. A second line of investigation will explore Ca2+-dependent changes in conformation at the A1-C2 junction regarding spatial changes in A1 domain relative to the membrane surface and Ca2+-dependent protease protection. Aim III studies will define a model for the exosite-dependent, thrombin-catalyzed activation of factor VIII following binding interactions using factor VIII chains and VIIIa subunits, as well as variant proteins constructed with point mutations at potentially critical exosite-interactive regions. Reciprocal binding and activity studies will use thrombin variants possessing point mutations in anion binding exosites I or II. A focal point of these studies is the rate-limiting cleavage at Arg372 which exposes cryptic factor IXa-sites in the cofactor. Additional studies will address the influence of P3-P3< residues on catalysis at Arg372. These aims will be facilitated by use of novel reagents including recombinant protease forms, substrates comprised of highly purified factor VIII chains (VIIIa subunits) and variants possessing point mutations at residues of interest. We will accomplish these studies using high-resolution techniques including fluorescence energy transfer, surface plasmon resonance and MALDI-TOF mass spectrometry. Results from these studies will define mechanisms providing significant insights into factor VIII structure and its regulation, as well as provide important information for the design of superior therapeutics for the treatment of hemophilia A.